The DNA of eukaryotes is associated with histones and nonhistone chromosomal proteins (NHC proteins) in a complex structure referred to as chromatin. We wish to determine in detail the differences in chromatin structure related to gene activity and to establish the macromolecular basis for these differences. In particular, we plan to test the hypothesis that certain chromatin structures which can be detected in terms of nuclease sensitivity are necessary for gene activation and transcription. Drosophila is the organism of choice for our work because of the facility with which biochemical, genetic, and cytological techniques can all be utilized. Using DNase I and micrococcal nuclease as probes, we plan to map the chromatin structure (position of DNase I hypersensitive sites, position of nucleosome sites, boundaries of regions of perturbation on gene activation) for several different types of loci, including a generally inducible locus (heat shock locus 67B), a developmentally regulated locus (vitellogenin), and a locus expressed at a low constant level (encoding a ribosomal protein). We are particularly interested in the role of NHC proteins in initiating and maintaining changes in chromatin structure relating to gene activity. We have identified several NHC proteins preferentially associated with either 1) the set of immediately active loci, or 2) the active loci and those loci known to be active at some time in the given cell type (salivary gland). Using antibodies to these proteins, we plant to test their role in maintaining particular chromatin structures and in maintaining transcriptional activity. We hope to use these proteins to fractionate chromatin by affinity chromatography, allowing further characterization of subsets of "active" nucleosomes. The information derived should allow us to begin to create structural models of chromatin that are relevant to the control of gene expression.